Browsing Department of Biochemistry by browse.metadata.advisor "Botha, F. C."
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- ItemSucrose accumulation and the expression of neutral invertase in sugarcane(Stellenbosch : Stellenbosch University, 2001-12) Rose, Susan, 1977-; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The goals of this project were to (i) determine maximum extractable neutral invertase (NI) activity in the sugarcane culm, (ii) sequence a cDNA encoding for the sugarcane NI (SNI), (iii) determine SNI copy number in the genome, (iv) describe SNI transcript and protein expression patterns throughout the plant, and (v) attempt to determine the contribution of hydrolysis to sucrose accumulation. SNI and sugars were extracted from the developing culm tissues of sugarcane, commercial variety N19. Tissues were divided according to developmental stage (internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular or storage tissues). The lowest sucrose content was found in the core of the bottom of each of the internodes. The ratio between hexoses and sucrose was highest in the young internodes. In these internodes hexose content was higher in the bottom than the top. There was a significant correlation between sucrose content and NI. Fluxes involved in sucrose synthesis and hydrolysis were investigated. The hexoses glucose and fructose were supplied as a carbon source for tissue discs of young and maturing internodal tissues of sugarcane, varieties N19 and US6656-15. Sucrose content was 10-fold higher in maturing internodes of N19 than US6656-15. Calculated sucrose hydrolysis rates via invertase were higher in maturing internodes of US6656-15 than N19. Taking metabolic compartmentation into account, hydrolysis of sucrose via invertase made a significant contribution to the net turnover of sucrose. Along with this, it would appear that the ability to partition sucrose between the vacuole and cytosol causes a significant difference in sucrose content between varieties. A full-length cDNA for SNI was sequenced. This expressed gene showed significant homology to known NI sequences on both nucleic and amino acid levels. The SNI sequence did not contain the putative invertase catalytic amino acid sequence, suggesting it developed separately from the other classes of invertases. Approximately 1.8 kb of the SNI cDNA was incorporated into a vector suitable for direct bombardment into sugarcane tissue. Southern blot analysis showed the enzyme has a low copy number. SNI transcript expression was observed in all tissues of the sugarcane plant: roots, internodes, leaf roll and leaves. In culm tissues where sucrose content was low and hexose contents were high, SNI transcript and protein levels were high. This suggests that SNI is involved in growth metabolism.